The primary objective of the proposed studies is the elucidation of the diverse mechanisms by which the neuropeptide substance P (SP) modulates immunologic responses through effects on human T-lymphocytes. SP is present at elevated concentrations in tissues at sites of inflammation and is known to influence the activities of several types of leukocytes. The initial demonstration by the principal investigator that SP stimulates the proliferation of purified human blood T-lymphocytes and cultured human IM-9 lymphocytes provides standardized systems for investigations of both the specificity and biochemical requirements of the regulation of T-lymphocyte functions by SP. The recent finding that IM-9 lymphocytes and a subset of T-lymphocytes express stereospecific receptors for SP indicate that analyses of the structural characteristics of the receptors will reveal the molecular bases for specific recognition of SP and the cellular events which SP initiates to activate T-lymphocytes. The specific aims of the proposal encompass an integrated assessment of the effects of SP on the functional responses of T-lymphocytes and IM-9 lymphocytes to antigens, mitogens, and other stimuli, the modulation by SP of the secretion and activity of lymphokines by T-lymphocytes, and the capacity of SP to influence T-lymphocyte regulation of B-lymphocyte function. T-lymphocyte responses will be assessed by the uptake of [3H]-thymidine and [3H]-leucine in proliferation assays, the measurement of I12-dependent proliferation of cytolytic T-lymphocytes, the quantification of leukocyte-inhibition factor generation, and sterile FACS isolation of subsets of T-lymphocytes which will be examined for proliferative and synthetic activities separately before and after exposure to SP. Fluorescent and solid-phase RIA measurements of immunoglobulins (Ig) and dual-color FACS analyses will be used to assess the effect of SP on T-lymphocyte regulation of B-cell Ig production and the expression of SP and Ig on the surface of IM-9 cells. The binding properties of the SP-receptor on IM-9 cells will be defined and the solubilized receptor will be isolated by chromatography on SP-affinity columns, and by chemical cross-linking of the receptors. Antibodies to the SP-receptor which may serve to elucidate its expression on other leukocytes will be developed both by immunizing goats and monoclonal techniques. An increased understanding of the recognition of SP by human T-lymphocytes and the immunological consequences of the interaction will elucidate an important aspect of the regulation of local tissue immunity by neuropeptides of the peripheral nervous system.